| Renal fibrosis is a common pathological feature of chronic kidney disease(CKD),which affects more than 10% of the global population and is a major public health problem.At present,TGF-β is the most critical fibrogenic factor,and it is closely related to the changes of metabolites in the process of fibrosis.In the TGF-β/Smad signaling pathway,Smad ubiquitin ligase Smurfs plays an important role in the regulation of TGF-β/Smad signaling pathway.Based on the previous research results,we found that Shenkang injection can achieve the effect of anti renal fibrosis by regulating the TGF-β/Smad signaling pathway,but its target of anti renal fibrosis and molecular pharmacological mechanism in this pathway are not very clear.Combined with relevant literature reports and our previous research,we proposed that "Shenkang injection may play an anti renal fibrosis role by promoting the degradation of TGF-β receptor TβR-I/-II and p-Smad2/3 in TGF-β/Smad signaling pathway induced by Smurfs".Therefore,the purpose of this experiment is to explore whether Shenkang injection can achieve anti renal fibrosis effect by promoting the ubiquitination degradation of TGF-β/Smad signaling pathway induced by Smurfs.Objective:1.Metabonomics technology was used to analyze the metabolites of ureteral ligation animal model intervened by Shenkang injection,to screen out the differential metabolites related to renal fibrosis,and to analyze the correlation between its metabolic pathway and TGF-β/Smad signaling pathway,which is helpful to explain the inhibitory mechanism of Shenkang Injection on renal fibrosis from the metabolic level and molecular level.2.By means of cell transfection,immunoprecipitation and subcellular co localization,it is clear that Shenkang injection can promote the degradation of TGF-β receptor TβR-I/II and p-Smad2/3 in the TGF-β/Smad signaling pathway induced by Smurfs and play the role of anti renal fibrosis.Method:1.Male C57BL/6J mice aged 6-8 weeks were selected as the research objects.The UUO model of renal interstitial fibrosis was established by ligation of the left ureter.Shenkang injection was used as the treatment drug.After 14 days of intervention,the materials were obtained after anesthesia.Enzyme linked immunosorbent assay(ELISA)was used to detect creatinine(CRE)and blood urea nitrogen(BUN),The expression of Smad7,TGF-β1,TβR-I,TβR-II,E-cad,α-SMA,Smurf1,Smurf2,p-smad3,Smad3,p-Smad2/3,Smad/3,Ubiquitin(Ub)protein in the tissues of mice were detected by immunohistochemistry;Western blot was used to detect the expression of Smad7,TGF-β1,TβR-I,TβR-II,E-cad,α-SMA,Smurf1,Smurf2,p-smad3,p-Smad2/3,Smad2/3,Ubiquitin(Ub)protein The protein expression of Smad7,TGF-β1,TβR-I,TβR-II,E-cad,α-SMA,Smurf1,Smurf2,p-smad3,Smad3,p-Smad2/3,Smad2/3,Ubiquitin(Ub)was detected by WB;the m RNA expression of Smad7,TGF-β1,TβR-I,TβR-II,E-cad,α-SMA,Smurf1,Smurf2 were detected by RT-PCR The binding degree of TβR-I-smad2,TβR-I-smad3,TβR-I-smurf2 and TβR-I-TβR-II in the kidney tissue of each group was detected by immunoprecipitation technique.2.Non targeted metabonomics analysis was performed by LC-MS/MS to study the serum samples of mice with renal interstitial fibrosis model and mice treated with Shenkang injection.The differential metabolites related to renal fibrosis were screened.The relationship between the differential metabolic pathway and TGF-β/Smad signaling pathway was analyzed by KEGG pathway enrichment.3.CCK-8 method was used to detect the effect of 1:10 to 1:100000 dilution of Shenkang Injection on HK-2 activity,so as to determine the safe concentration range of Shenkang Injection in HK-2 cells.Three concentrations were selected as the experimental concentration for subsequent intervention of HK-2 cells.4.TGF-β1 was used to induce HK-2 cell transdifferentiation(EMT)and Shenkang injection was given to intervene.The condition of EMT was observed by inverted microscope.The expression and localization of Smurf1/2-smad7,smurf1-TβR-I and smurf2-smad2 in EMT(α-SMA,E-cadherin)markers and TGF-β/Smad signal molecules were detected by immunofluorescence and inverted fluorescence microscope The protein expression of Smad7,TGF-β1,TβR-I,TβR-II,E-cad,α-SMA,Smurf1,Smurf2,p-smad3,Smad3,p-Smad2/3,Smad2/3,Ubiquitin(Ub)was analyzed by WB;the m RNA expression of Smad7,TGF-β1,TβR-I,TβR-II,E-cad,α-SMA,Smurf1,Smurf2 was detected by RT-PCR.5.Myc-TβR-I or TβR-II,Flag-Smurf1 and HA-Ub were cotransfected into HK-2 cells by plasmid transfection method.At the same time,TGF-β1 was stimulated and Shenkang injection was given for intervention.The expression and binding of TβR-I,TβR-II,Smurf1 and Ub proteins were observed and detected by immunoprecipitation and Western blot.6.Flag-smurf2,Myc-smad2 and HA-Ub were cotransfected into HK-2 cells by plasmid transfection.TGF-β1 and Shenkang injection were used to stimulate the transfection.The expression and binding of Smurf2,Ub and Smad2 proteins were detected by immunoprecipitation and Western blot.7.The interaction between TβR-I or TβR-II and Smad2 or Smad3 was observed by immunoprecipitation assay.Result:1.Shenkang injection can improve the renal function of UUO model mice and promote the recovery of body weight;through he and Masson staining on the affected side of the kidney,the results showed that Shenkang injection can reduce the atrophy and expansion of renal tubules,the reduction of the number of renal tubules,cystic expansion of renal tubular epithelial cell apoptosis,inflammatory cell infiltration and the area of renal interstitial fibrosis,reduce the synthesis of Co L-I and col-iii,and inhibit the apoptosis of renal tubules The expression of α-SMA and E-cad were increased.In immunohistochemistry,Western blot and RT-PCR experiments,Shenkang injection decreased the expression of TβR-I,TβR-II,Smad3,Smad2 and p-Smad2/3 in UUO model mice,promoted the expression of Smad7,Smurf2,Smurf1 and Ubiquitin,and inhibited the binding of TβR-I-smad2,TβR-I-Smad3,TβR-ISmurf2 and TβR-I-TβR-II.2.The key pathways of Shenkang Injection on serum metabolites of UUO model mice are bile secretion pathway,metabolism of vitamin B6,metabolism of taurine and low taurine,steroid metabolism,phenylalanine metabolism,phosphoinositide metabolism,steroid hormone biosynthesis,purine metabolism,alanine,tyrosine,glutamate and aspartate metabolism,pyruvate metabolism,nicotinic acid metabolism Shenkang injection can restore the disorder of lipid metabolism,amino acid metabolism and purine metabolism in UUO model mice by negatively regulating TGF-β/Smad signaling pathway.3.The effects of Shenkang Injection on the survival rate of HK-2 cells were 14.92%,13.12%,91.78%,110.62%,107.11%,105.39%,97.48%,104.31%,109.07% and109.46% respectively.Therefore,1:40,1:80 and 1:100 were selected as the high,medium and low concentrations of Shenkang Injection(SKI-H,SKI-M and SKI-L).The m RNA expressions of TGF-β1,TβR-I and TβR-II in normal HK-2 cells treated with high,medium and low concentrations of Shenkang injection decreased,while the m RNA expressions of Smad7,Smurf1 and Smurf2 increased.The results showed that Shenkang injection could reduce the expression of α-SMA protein,increase the expression of E-cad protein,inhibit EMT of HK-2 cells,reduce the expression of TβR-I,TβR-II,Smad3,Smad2,p-Smad2/3,and promote the expression of Smad7,Smurf2,Smurf1,Ubiquitin in HK-2 cells treated with TGF-β1.4.HK-2 cells treated with Shenkang injection significantly inhibited the binding of TβR-I-TβR-II,TβR-I/II-Smad2,TβR-I/II-Smad3 and TβR-I/II-Smad2/3.5.Shenkang injection can increase the fluorescence intensity of Smurf1,Smurf2 and Smad7 in HK-2 cells induced by TGF-β1,and promote the nuclear translocation of Smurf1-Smad7 and Smurf2-Smad7.6.In HK-2 cells overexpressing Myc-TβR-I,Flag-Smurf1,HA-Ub and Myc-Smad2,Flag-Smurf2,HA-Ub,Shenkang Injection promoted the binding of Smad ubiquitin ligases Smurf2-Smad2 and Smurf1-TβR-I,and made TβR-I and Smad2 ubiquitinated and degraded by Smurf1 and Smurf2,respectively. |
PDF Full Text Request